Huntington's disease (HD) is a progressive fatal neurodegenerative disease caused by expansion in a polyglutamine encoding CAG tract in the huntingtin gene. The normal function of the huntingtin protein is not well understood. The precise nature of polyglutamine toxicity and the key targets that it acts upon to cause cellular dysfunction also remain to be elucidated. In order to provide greater insight into the normal and pathogenic functions of huntingtin we undertook a large-scale screen to discover huntingtin interacting proteins (HIPs) using yeast two-hybrid and mass spectrometry-based methods. After data analysis using numerical and statistical methods, a high confidence group of 234 HIPs was identified. In order to test these for relevance to the HD pathology, genes encoding orthologs of 60 interacting proteins were tested for their ability to modify a toxic polyglutamine phenotype in a Drosophila model of HD. 80% of those genes tested acted as modifiers of the HD toxicity indicating that the ability of a protein to physically interact with huntingtin correlated with its ability to show genetic interaction in a phenotypic assay. Genetic analysis in Drosophila has thus far identified 25 loss-of-function suppressors of HD toxicity. These results demonstrate that the ensemble of HIPs identified in our study are enriched for proteins that play a direct role in the cellular pathology of HD. These results also suggest that HIPs may be similarly enriched for proteins that can modify HD in human. The primary goal of this study is to test HIPs identified in our high-throughput studies for their ability modify HD phenotypes in mammalian cells and HD mouse models. We will use siRNAs to knock-down expression of each of these genes in human and mouse neuronal cell models of mutant HD toxicity. In specific cases, gene expression knock-downs will also be evaluated for effects on the metabolism, localization and/or post-translational modification of the huntingtin protein. Candidate proteins showing effects in these assays will be prioritized for more extensive studies in mouse models of HD. HIPs that modify the toxicity and/or biochemical properties of huntingtin will be tested for co-localization with huntingtin in transgenic HD mouse brain. HIPs whose reduced expression can suppress toxicity in cell-based assays (and/or Drosophila) will be tested for suppression in mouse models of HD. This will be done by constructing the appropriate shRNA-expressing transgenic mouse lines, crossing these into HD mouse models, and studying effects on the mouse HD phenotypes. We will also use AAV-mediated viral transfection of HIP shRNA to study effects of HIP knock-down in HD mice. The ultimate purpose of these studies is to identify modifiers of HD phenotypes in cell-based and/or Drosophila assays, determine their mechanisms of action and validate these in mouse models of HD. We anticipate that these studies will provide useful insight into the nature of HD pathology and also provide novel candidate targets for therapeutic drug discovery in HD.